Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
  • C12N9/20—Triglyceride splitting, e.g. by means of lipase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
  • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)

Definitions

• the present invention relates to a method of preparing cross-linked enzyme aggregates comprising the steps of a) precipitating a dissolved enzyme present in a liquid medium; b) cross-linking with a cross-linker, and c) optionally, washing the cross-linked enzyme aggregate
• Such a method is generally known in the art. It is used to prepare enzyme preparations for performing various enzyme-catalysed reactions.
• Cao L. et al. (Or- ganic Letters vol. 2 (10) p. 1361-1364, 2000) describe the preparation of penicilin acylase-based aggregates.
• the cross- linked aggregates disclosed are less active than the native enzyme.
• the object of the present invention is to provide a method making it possible to achieve aggregates with increased enzymatic activity.
• the invention is characterized in that at least one treatment is performed chosen from the group consisting of I) performing step a) in the presence of a com- pound selected from the group consisting of i) a crown ether, and ii) a surfactant, and II) performing step c) with a second liquid differing in organic composition from the liquid.
• Applicants have surprisingly found that such a treatment may result in an cross-linked enzyme aggregate (CLEA) with an enhanced activity. While the present invention does neither guarantee this enhanced activity for a particular enzyme used to catalyse a particular reaction, nor allows for a prediction of an enhanced activity, it does provide for an extra parameter to be controlled to achieve a better ag- gregate. That is, for a particular enzyme, it may be investigated with little effort and using simple and routine experimentation whether its catalytic activity can be enhanced using the treatment. From a range of enzymes capable of catalyzing the desired reaction, the best CLEA can easily and with little effort be selected.
• CLSA cross-linked enzyme aggregate
• the CLEA is insoluble, it can be washed with a second liquid which is com- pletely aqueous, organic, or a mixture of the two. After the treatment, the CLEA may be lyophilized or stored as a suspension for future use.
• step a) and step b) are performed simultaneously.
• the compound is removed after cross-linking.
• the surfactant and or crown ether are present during the catalytic reaction to be performed by the CLEA to maintain the high activity.
• the compound may be removed, in case it may interfere with the reaction to be catalysed, or for other reasons. Removal may be achieved using any method known in the art, and in particular through dialysis or washing using centrifugation.
• the enzyme is a lipase.
• the invention also relates to a method for performing a lipase-catalysed reaction, said reaction being chosen from the group consisting of i) transesterification; ii) interesterification; iii) hydrolysis; iv) esterification; v) ammoniolysis; vi) aminolysis; and vii) perhydrolysis, which method is characterized, in that a cross-linked lipase- aggregate is prepared according to the invention, and said cross-linked lipase-aggregate is contacted with a substrate.
• JR. iehei lipase Lipozyme (Novo Nordisk) Candida antarctica lipase B SP525 (Novo Nordisk) P. alcaligenes lipase (Gist-brocades) .
• METHOD la In the presence of (NH 4 ) 2SO 4 (Wa ter) 0.5 ml stock enzyme solution (Lipozyme or SP525, Novo Nor- disk, Copenhagen, Denmark) or 50 mg PaL enzyme powder (PaL is P. alcaligenes lipase (Gist-Brocades, Delft, the Netherlands) ) , are dissolved in 1 ml potassium phosphate buffer (100 mM, pH 7) in a 10 ml centrifuge tube.
• 550 mg (NH 4 ) 2 S0 was added followed by 1 ml of a precipitating solution A con- sisting of 550 mg (NH) 2 S0 (the precipitant) in potassium phosphate buffer (100 mM, pH 7), and 80 ⁇ l glutardialdehyde (25% solution in water) are added. The mixture is left stirring at 4°C (room temperature for SP525) for 17 hours. 3 ml H 2 0 is added to the mixture and then centrifuged. The su- pernatant is decanted and the residue is washed, centrifuged and decanted 3 more times with H 2 0 (5 ml each time) . The final enzyme preparation is kept in 5 ml H0.
• aqueous suspensions 0.5 ml samples of CLEA suspension were lyophilized in 2 ml Eppendorf cup each.
• organic suspensions 0.5 ml samples of CLEA suspension were centrifuged in a 2 ml Eppendorf cup each, and the supernatant was decanted.
• the enzyme preparation (50 ⁇ l) was suspended in 0.5 ml water (1.5 ml in case of SP525) and 200 ⁇ l (50 ⁇ l in case of SP525) of the suspension was added to 2.5 ml ester solution (0.4 mM p-nitrophenyl propionate in water at 25°C) .
• adding a surfactant or a crown ether in accordance with the present invention may have a beneficial effect on the enzymatic activity.
• Methode la was repeated using SP525.
• the supernatant is decanted and the pellet resuspended in 5 ml water, centrifuged and the supernatant is decanted.
• the pellet obtained is subjected to the same washing procedure with 3 * 5 ml water or DME.
• the CLEA is kept in 5 ml of the same solvent.
• the two CLEAs were tested using the transesterification of ethyl octanoate with octanol (as described in test lie) .
• 0.5 ml DME is added to 50 ⁇ l enzyme preparation in a 2 ml Eppendorf. The enzyme washed with water only was previously ly- ophilized in the Eppendorf cup.
• Lipase CLEAs were prepared according the procedure described in example 1 and were tested in the hydrolysis of methyl mandelate. The enzyme was added to a 50 mM solution of methyl mandelate in dimethoxyethane-potassium phosphate buffer pH 7 (95:5, v/v) . After 48 hours the conversion and enantiomeric excess of the substrate (ee s ) were analyzed using HPLC (see Table 5) .

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• Enzymes And Modification Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
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• 2001
  • 2001-02-01 NL NL1017258A patent/NL1017258C2/nl not_active IP Right Cessation
• 2002
  • 2002-02-01 EP EP02711535A patent/EP1360285B1/de not_active Expired - Lifetime
  • 2002-02-01 CA CA002436087A patent/CA2436087A1/en not_active Abandoned
  • 2002-02-01 WO PCT/NL2002/000079 patent/WO2002061067A1/en not_active Ceased
  • 2002-02-01 US US10/470,895 patent/US20040235126A1/en not_active Abandoned
  • 2002-02-01 JP JP2002561624A patent/JP2004520050A/ja active Pending
  • 2002-02-01 AT AT02711535T patent/ATE428776T1/de not_active IP Right Cessation
  • 2002-02-01 PT PT02711535T patent/PT1360285E/pt unknown
  • 2002-02-01 DE DE60231959T patent/DE60231959D1/de not_active Expired - Fee Related
  • 2002-02-01 ES ES02711535T patent/ES2323572T3/es not_active Expired - Lifetime
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